Pharmaceutical compounds

ABSTRACT

Therapeutically-effective amounts of novel analogs or derivatives of alkyl fatty acids, such as but not limited to lipoic acid, and pharmaceutical formulations comprising such analogs or derivatives and pharmaceutically-acceptable carriers therefor, are useful for the treatment, prevention, imaging, and/or diagnosis of medical disorders.

FIELD OF THE INVENTION

This invention relates to pharmaceutical agents, and more particularlyto therapeutic agents comprising novel analogs and derivatives of alkylfatty acids, such as but not limited to lipoic acid, andpharmaceutically-acceptable formulations and methods of use therefor.

BACKGROUND OF THE INVENTION

The precise mechanism by which cancer arises continues to be the subjectof intense investigation, and thus a unifying theory of the origin ofcancer remains elusive. Recent research has confirmed that cancer is adisease arising from a patient's own cells and tissue. Indeed, it is nowknown that an individual patient may possess multiple tumor cell types,which may not be the same across patients with the same diagnosis oreven in the same patient (with disease progression being a furthercompounding factor). In any event, the highly individualized nature ofthe disease is an important factor in driving the need for personalizedmedicine. That 1.2 million Americans are newly diagnosed each year withcancer; that 10 million Americans are living with the disease; and thatcancer may become the leading cause of disease-related death makes theestablishment of new treatment approaches especially urgent.

It has been observed that the vast majority of fast-growth tumor cellsexhibits profound genetic, biochemical, and histological differenceswith respect to nontransformed cells, including a markedly-modifiedenergy metabolism in comparison to the tissue of origin. The mostnotorious and well-known energy metabolism alteration in tumor cells isan increased glycolytic capacity even in the presence of a high O₂concentration, a phenomenon known as the Warburg effect. Consequently,glycolysis generally believed to be the main energy pathway in solidtumors. There is also a direct correlation between tumor progression andthe activities of the glycolytic enzymes hexokinase andphosphofructokinase (PFK) 1, which are greatly increased in fast-growthtumor cells. Accordingly, it has been postulated that tumor cells thatexhibit deficiencies in their oxidative capacity are more malignant thanthose that have an active oxidative phosphorylation. No matter whetherunder hypoxic or aerobic conditions, then, cancer tissue's reliance onglycolysis is associated with increased malignancy.

The pyruvate dehydrogenase (PDH) complex has been associated with theWarburg effect. (See, e.g., McFate T, Mohyeldin A, Lu H, Thakar J,Henriques J, Halim N D, Wu H, Schell M J, Tsang T M, Teahan O, Zhou S,Califano J A, Jeoung N H, Harris R A, and Verma A (2008). Pyruvatedehydrogenase complex activity controls metabolic and malignantphenotype in cancer cells. J Biol Chem 283:22700-8, herein incorporatedby reference.) The transition to Warburg metabolism therefore requiresshutting down the PDH complex. In this transition, there is enhancedsignalling by hypoxia-inducing factor (HIF) in cancer cells, which inturn induces the overexpression of pyruvate dehydrogenase kinase (PDK)1, which is particularly effective in maintaining an inactive PDHcomplex. However, alterations in PDK1 observed in cancer may not only bedue to changes in its concentration but also to changes in its activityand possibly in its amino acid sequence, even between one tumor type orone patient to another. Additionally, PDK1 may form different complexeswith various molecules associated with tumors depending upon the tumortype presented. Recent studies suggest that forcing cancer cells intomore aerobic metabolism suppresses tumor growth. Furthermore, PDHcomplex activation may lead to the enhanced production of reactiveoxygen and nitrogen species (RONS), which may in turn lead to apoptosis.Thus, inhibition of PDK may be a potential target in generatingapoptosis in tumors. However, to date, known PDK1 inhibitors have beendemonstrated to cause maximally only 60% inhibition of this isozyme.

While traditional chemotherapy targets dividing, proliferating cells,all clinically-accepted chemotherapeutic treatments use large drug dosesthat also induce profound damage to normal, proliferative host cells. Onthe other hand, drug delivery to a hypoxic region in solid tumors may bedifficult when the drug does not permeate through the different cellularlayers easily. Therefore, more selective targeting is required for thetreatment of cancer. Another problem associated with chemotherapy isthat, in many tumor types, there is either inherent or acquiredresistance to antineoplastic drugs. Overall, traditional chemotherapycurrently offers little long-term benefit for most malignant tumors andis often associated with adverse side-effects that diminish the lengthor quality of life.

Hence, radical new approaches are required that can provide long-termmanagement of tumors while permitting a decent quality of life. Tofulfill these imperatives, it would be advantageous to design anticanceragents having metabolic inhibition constants in at least thesubmicromolar range. Concentrating on the Warburg effect allows fordesigning drugs based on the physico- and biochemical energeticdifferences between tumor and normal cells to facilitate the design ofdelivery and therapeutic strategies that selectively affect solely tumormetabolism and growth without affecting healthy tissue function.

Lipoic acid (6,8-dithiooctanoic acid) is a sulfur-containing antioxidantwith metal-chelating and anti-glycation capabilities. Lipoic acid is theoxidized part of a redox pair, capable of being reduced to dihydrolipoicacid (DHLA). Unlike many antioxidants that are active only in either thelipid or the aqueous phase, lipoic acid is active in both lipid andaqueous phases. The anti-glycation capacity of lipoic acid combined withits capacity for hydrophobic binding enables lipoic acid to preventglycosylation of albumin in the bloodstream. Lipoic acid is readilyabsorbed from the diet and is rapidly converted to DHLA by NADH or NADPHin most tissues. Additionally, both lipoic acid and DHLA areantioxidants capable of modulating intracellular signal transductionpathways that use RONS as signalling molecules.

It is uncertain whether lipoic acid is produced by cells or is anessential nutrient, as differences in intracellular concentration mayexist between tissue types as well as between healthy and diseased cellsor even between individuals within a species. Mitochondrial pumps oruptake mechanisms, including binding and transport chaperones, may beimportant in transporting lipoic acid to mitochondria. It is alreadyknown that the expression levels and stoichiometry of the subunitscomprising many of the lipoic acid-utilizing enzymes, which are linkedto energy metabolism as well as growth, development and differentiation,vary with diet and exercise as well as genetics. The role of lipoic acidas a cofactor in the PDH complex of healthy cells has been well studied.The PDH complex has a central E2 (dihydrolipoyl transacetylase) subunitcore surrounded by the E1 (pyruvate dehydrogenase) and E3 (dihydrolipoyldehydrogenase) subunits to form the complex; the analogousalpha-ketoglutarate dehydrogenase (α-KDH), acetoin dehydrogenase (ADH),and branched chain alpha-keto acid dehydrogenase (BCKADH) complexes alsouse lipoic acid as a cofactor. In the gap between the E1 and E3subunits, the lipoyl domain ferries intermediates between the activesites. The lipoyl domain itself is attached to the E2 core by a flexiblelinker. Upon formation of a hemithioacetal by the reaction of pyruvateand thiamine pyrophosphate, this anion attacks the S1 of an oxidizedlipoate species that is attached to a lysine residue. Consequently, thelipoate S2 is displaced as a sulfide or sulfhydryl moiety, andsubsequent collapse of the tetrahedral hemithioacetal ejects thiazole,releasing the TPP cofactor and generating a thioacetate on the S1 of thelipoate. At this point, the lipoate-thioester functionality istranslocated into the E2 active site, where a transacylation reactiontransfers the acetyl from the “swinging arm” of lipoate to the thiol ofcoenzyme A. This produces acetyl-CoA, which is released from the enzymecomplex and subsequently enters the TCA cycle. The dihydrolipoate, stillbound to a lysine residue of the complex, then migrates to the E3 activesite, where it undergoes a flavin-mediated oxidation back to its lipoateresting state, producing FADH₂ (and ultimately NADH) and regeneratingthe lipoate back into a competent acyl acceptor.

U.S. Pat. Nos. 6,331,559 and 6,951,887 to Bingham et al., as well asU.S. patent application Ser. No. 12/105,096 by Bingham et al., allherein incorporated by reference, disclose a novel class of lipoic acidderivative therapeutic agents that selectively target and kill bothtumor cells and certain other types of diseased cells through targetingdisease-specific enzymes and multi-enzyme complexes. These patentsfurther disclose pharmaceutical compositions, and methods of usethereof, comprising a therapeutically-effective amount of such lipoicacid derivatives along with a pharmaceutically-acceptable carriertherefor. The present inventors have now discovered additional analogsand derivatives beyond the scope of the aforementioned patents.

SUMMARY OF THE INVENTION

The present invention is directed to an analog or derivative of an alkylfatty acid, such as but not limited to lipoic acid, having the generalformula:

wherein n is 1-2 and x is 1-16, with the resulting hydrocarbon chainpotentially being mixed saturated or unsaturated;

wherein R₁ and R₂ are independently hydrogen, alkyl, alkenyl, alkynyl,alkylaryl, heteroaryl, or alkylheteroaryl;

wherein R₃ and R₄ are independently a thioether, a thioester, an ether,an ester, an amine, an amide, a rare earth metal such as but not limitedto gadolinium, a transition metal such as but not limited to platinum orindium, or a nonmetal such as but not limited to selenium;

wherein R₅ is hydrogen, nitrogen, alkyl, alkenyl, alkynyl, alkylaryl,heteroaryl, alkylheteroaryl, an ether, an amine, an amide, aphosphonium, a phosphonate, or a sulfonic acid;

and salts, prodrugs, or solvates thereof.

Specific examples are provided hereinbelow.

In a further embodiment of the present invention, atherapeutically-effective amount of at least one alkyl fatty acid analogor derivative as described herein is combined with at least onepharmaceutically-acceptable carrier or excipient therefor to form apharmaceutical formulation useful for the treatment, prevention,imaging, or diagnosis of a disease of warm-blooded animals, includinghumans, wherein diseased cells or tissue are sensitive to such alkylfatty acid analogs or derivatives. The at least one alkyl fatty acidanalog or derivative is present in an amount from about 0.001 mg/m² toabout 10 g/m². Additionally, as any or all of these analogs orderivatives may be metabolized within the diseased cell, ormitochondrion or other organelle thereof, it is expressly intended thatmetabolites of the above-referenced analogs or derivatives be within thescope of the present invention. Furthermore, in each of the generalformulae, the (R)-isomer of each particular compound possesses greaterphysiological activity than does the (S)-isomer. Consequently, the atleast one analog or derivative should be administered either solely inthe (R)-isomer form or in a mixture of the (R)- and (S)-isomers.

In a still further embodiment of the present invention, there isprovided a method of treating, preventing, imaging, or diagnosing adisease characterized by disease cells or tissue of warm-bloodedanimals, including humans, that are sensitive to administration of analkyl fatty acid analog or derivative as described herein, comprisingadministering to a patient in need thereof a therapeutically-effectiveamount of at least one alkyl fatty acid analog or derivative accordingto any of the embodiments of the invention. In a preferred embodiment,the at least one alkyl fatty acid analog or derivative is combined withat least one pharmaceutically-acceptable carrier therefor to form apharmaceutical formulation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to novel analogs or derivatives of analkyl fatty acid, such as but not limited to lipoic acid, having thegeneral formula:

wherein n is 1-2 and x is 1-16, with the resulting hydrocarbon chainpotentially being mixed saturated or unsaturated;

wherein R₁ and R₂ are independently hydrogen, alkyl, alkenyl, alkynyl,alkylaryl, heteroaryl, or alkylheteroaryl;

wherein R₃ and R₄ are independently a thioether, a thioester, an ether,an ester, an amine, an amide, a rare earth metal such as but not limitedto gadolinium, a transition metal such as but not limited to platinum orindium, or a nonmetal such as but not limited to selenium;

wherein R₅ is alkyl, alkenyl, alkynyl, alkylaryl, heteroaryl, oralkylheteroaryl;

wherein R₆ is hydrogen, nitrogen, alkyl, alkenyl, alkynyl, alkylaryl,heteroaryl, alkylheteroaryl, an ether, an amine, an amide, aphosphonium, a phosphonate, or a sulfonic acid;

and salts, prodrugs, or solvates thereof.

Particular alkyl fatty acid analogs or derivatives according to generalformula (1) include:

As used herein, alkyl is defined as C_(n)H_(2n+1), wherein n is 1-16.Alkyl groups can be either aliphatic (straight or branched chain) oralicyclic; alicyclic groups may have additions or substitutions on anyof the carbons to form heterocyclics. At least one heteroatom such as N,O or S may be present in a given alkyl group, i.e., in the carbon chain.Alkyl groups may be substituted or unsubstituted on any of theircarbons.

As used herein, alkenyl is defined as C_(n)H_(2n−1), wherein n is 1-16.Alkenyl groups can be either aliphatic (straight or branched chain) oralicyclic; alicyclic groups may have additions or substitutions on anyof the carbons to form heterocyclics. At least one heteroatom such as N,O or S may be present in a given alkenyl group, i.e., in the carbonchain. Alkenyl groups may be substituted or unsubstituted on any oftheir carbons.

As used herein, alkynyl is defined as C_(m)H_(2m−3), where m is 2-10.Alkynyl groups can be either aliphatic (straight or branched chain) oralicyclic; alicyclic groups may have additions or substitutions on anyof the carbons to form heterocyclics. At least one heteroatom such as N,O or S may be present in a given alkynyl group, i.e., in the carbonchain. Alkynyl groups may be substituted or unsubstituted on any oftheir carbons.

As used herein, aryl refers to any univalent organic radical derivedfrom an aromatic hydrocarbon by removing a hydrogen atom. Aryl ispreferably an unsaturated ring system having 5-10 carbon atoms. Arylalso includes organometallic aryl groups such as ferrocene. Aryl groupsmay be substituted or unsubstituted on any of their carbons.

As used herein, heteroaryl refers to an aromatic heterocyclic ringsystem (monocyclic or bicyclic) where the heteroaryl moieties are five-or six-membered rings containing 1-4 heteroatoms selected from the groupconsisting of S, N, and O. Heteroaryl groups may be substituted orunsubstituted on any of their atoms especially on the carbon atoms.

As used herein, acyl is defined as RC(O)—, where R can be, withoutlimitation, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl,alkylaryl, heteroaryl, or heterocyclyl, any of which can be substitutedor unsubstituted.

Exemplary substituents for the above-described groups include, withoutlimitation, alkyl, alkenyl, alkynyl, aryl, heteroaryl, acyl,alkoxycarbonyl, alkoxy, alkoxyalkyl, alkoxyalkoxy, cyano, halogen,hydroxy, nitro, oxo, trifluoromethyl, trifluoromethoxy, trifluoropropyl,amino, amido, alkylamino, dialkylamino, dialkylaminoalkyl, hydroxyalkyl,alkoxyalkyl, alkylthio, —SO₃H, —SO₂NH₂, —SO₂NH(alkyl), —SO₂N(alkyl)₂,—CO₂H, CO₂NH₂, CO₂NH(alkyl), and —CO₂N(alkyl)₂. In addition, any numberof substitutions may be made on any of the above-described groups; inother words, it is possible to have a mono-, di-, tri-, etc. substitutedgroup, and the substituents themselves may also be substituted. Further,any of the groups may be appropriately generally substituted with any ofa carbohydrate, a lipid, a nucleic acid, an amino acid, or a polymer ofany of those, or a single or branched chain synthetic polymer (having amolecular weight ranging from about 350 to about 40,000).

Amines may be primary, secondary, or tertiary.

Thioester or thioether linkages can be oxidized to produce sulfoxides orsulfones; in other words, the —S— in the linkage could be —S(O)— or—S(O)₂. In addition, thioester or thioether linkages may furthercomprise disulfides that can be oxidized to thiosulfinic or thiosulfonicacids; in other words, instead of —S— in a linkage, the linkage could be—S(O)—S— or —S(O)₂—S—.

A therapeutically-effective amount of at least one alkyl fatty acidanalog or derivative of any one of the aforementioned embodiments may beadministered to a subject for the treatment, prevention, diagnosis,and/or imaging of a disease, or symptoms thereof, in warm-bloodedanimals. Alternatively, in another embodiment of the present invention,a therapeutically-effective amount of at least one alkyl fatty acidanalogs or derivative of any one of the aforementioned embodiments iscombined with at least one pharmaceutically-acceptable carrier orexcipient therefor to form a pharmaceutical formulation useful for thetreatment, prevention, diagnosis, and/or imaging of a disease, orsymptoms thereof, in warm-blooded animals. Such animals include those ofthe mammalian class, such as humans, horses, cattle, domestic animalsincluding dogs and cats, and the like. Examples ofpharmaceutically-acceptable carriers are well known in the art andinclude those conventionally used in pharmaceutical compositions, suchas, but not limited to, solvents, diluents, surfactants, solubilizers,salts, antioxidants, buffers, chelating agents, flavorants, colorants,preservatives, absorption promoters to enhance bioavailability,antimicrobial agents, and combinations thereof, optionally incombination with other therapeutic ingredients. When used in medicine,the salts should be pharmaceutically acceptable, butnon-pharmaceutically-acceptable salts may conveniently be used toprepare pharmaceutically-acceptable salts thereof and are not excludedfrom the scope of the invention. Such pharmacologically- andpharmaceutically-acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, maleic, acetic, palicylic, p-toluene sulfonic,tartaric, citric, methane sulfonic, formic, malonic, succinic,naphthalene-2-sulfonic, and benzene sulfonic. Also,pharmaceutically-acceptable salts can be prepared as alkaline metal oralkaline earth salts, such as sodium, potassium or calcium salts of thecarboxylic acid group.

Solvents particularly suitable for use herein include benzyl alcohol,dimethylamine, isopropyl alcohol and combinations thereof; one ofordinary skill in the art would readily recognize that it may bedesirable to first dissolve the at least one lipoic acid derivative in asuitable solvent and then to dilute the solution with a diluent.

When a pharmaceutical formulation suitable for intravenousadministration is desired, a suitable diluent would be employed. Anyconventional aqueous or polar aprotic solvent is suitable for use in thepresent invention. Suitable pharmaceutically acceptable diluentsinclude, without limitation, saline, a sugar solution, alcohols such asethyl alcohol, methanol and isopropyl alcohol, polar aprotic solventssuch as dimethylformamide (DMF), dimethylsulfoxide (DMSO) anddimethylacetamide (DMA), and combinations thereof. A preferredpharmaceutically acceptable diluent is a dextrose solution, morepreferably a dextrose solution containing from about 2.5% to about 10%,more preferably about 5%, dextrose by weight. The pharmaceuticallyacceptable diluent is typically employed in a non-homolysis generatingamount; one of ordinary skill in the art can readily determine an amountof diluent suitable for use in a pharmaceutical formulation according tothe present invention.

As used herein, a therapeutically-effective amount refers to the dosageor multiple dosages of the alkyl fatty acid analog or derivative atwhich the desired effect is achieved. Generally, an effective amount ofthe analog or derivative may vary with the activity of the specificagent employed; the metabolic stability and length of action of thatagent; the species, age, body weight, general health, dietary status,sex and diet of the subject; the mode and time of administration; rateof excretion; drug combination, if any; and extent of presentationand/or severity of the particular condition being treated. The precisedosage can be determined by an artisan of ordinary skill in the artwithout undue experimentation, in one or several administrations perday, to yield the desired results, and the dosage may be adjusted by theindividual practitioner to achieve a desired effect or in the event ofany complication.

The alkyl fatty acid analog or derivative of the present invention canbe delivered, by any means, in any amount desired up to the maximumamount that can be administered safely to a patient. The amount of theanalog or derivative may range from less than 0.01 mg/mL to greater than1000 mg/mL, preferably about 50 mg/mL.

Generally, the alkyl fatty acid analog or derivative of the presentinvention will be delivered in a manner sufficient to administer to thepatient an amount effective to deliver the agent to its intendedmolecular target. The dosage amount may thus range from about 0.001mg/m² to about 10 g/m², preferably about 60 mg/m². The dosage amount maybe administered in a single dose or in the form of individual divideddoses, such as from one to four or more times per day. In the event thatthe response in a subject is insufficient at a certain dose, even higherdoses (or effective higher doses by a different, more localized deliveryroute) may be employed to the extent of patient tolerance.

As any or all of these analogs or derivatives may be metabolized withinthe diseased cell, or mitochondrion or other organelle thereof, uponadministration to the patient, it is expressly intended that metabolitesof the above-referenced analogs or derivatives be within the scope ofthe present invention. Furthermore, in each of the general formulae, the(R)-isomer of each particular compound possesses greater physiologicalactivity than does the (S)-isomer. Consequently, the at least one analogor derivative should be administered either solely in the (R)-isomerform or in a mixture of the (R)- and (S)-isomers.

The pharmaceutical formulation of the present invention can be preparedaccording to conventional formulation techniques and may take anypharmaceutical form recognizable to the skilled artisan as beingsuitable. Suitable pharmaceutical forms include solid, semisolid,liquid, or lyophilized formulations, such as tablets, powders, capsules,suppositories, suspensions, liposomes, emulsions, nanoemulsions,aerosols, sprays, gels, lotions, creams, ointments, and the like. Ifsuch a formulation is desired, other additives well-known in the art maybe included to impart the desired consistency and other properties tothe formulation. For example, a stock solution of the at least one alkylfatty acid analog or derivative can be prepared according toconventional techniques and then diluted as desired by apharmaceutically-acceptable diluent to form a liquid preparation such asa sterile parenteral solution.

The pharmaceutical formulation of the present invention may beadministered using any mode of administration both that is medicallyacceptable and that produces effective levels of the agent withoutcausing clinically-unacceptable adverse effects. Although formulationsspecifically suited for parenteral administration are preferred, thepharmaceutical formulation of the present invention may be contained inany suitable vessel, such as a vial or ampoule, and suitable for via oneof several routes including inhalational, oral, topical, transdermal,nasal, ocular, pulmonary, rectal, transmucosal, intravenous,intramuscular, intradermal, subcutaneous, intraperitoneal,intrathoracic, intrapleural, intrauterine, intratumoral, or infusionmethodologies or administration, without limitation. Those skilled inthe art will recognize that the mode of administering the analog orderivative of the present invention depends on the type of disease orsymptom to be treated. Likewise, those skilled in the art will alsorecognize that particular pharmaceutically-acceptable carriers orexcipients will vary from pharmaceutical formulations suitable for oneadministration mode to those suitable for another administration mode.

In a further embodiment of the present invention, there is provided amethod of treating, preventing, imaging, and/or diagnosing a diseasecharacterized by diseased cells or tissue that are sensitive to alkylfatty acid analogs or derivatives according to the present invention,comprising administering to a patient in need thereof atherapeutically-effective amount of at least one such analog orderivative. In a preferred embodiment, the at least one alkyl fatty acidanalog or derivative is incorporated into a pharmaceutical formulationaccording to the present invention.

The alkyl fatty acid analogs or derivatives of the present invention,and pharmaceutical formulations thereof, may be used to treat, prevent,image, or diagnose diseases involving altered or distinct cellular PDH,α-KDH, ADH, and/or BCKADH complex activity. Cells with altered orderanged PDH, α-KDH, ADH, and/or BCKADH complex activity areparticularly targeted, so that upon administration, the analog orderivative of the present invention is selectively and specificallydelivered to and taken up by a tumor mass and the transformed cellswithin, and effectively concentrated within the mitochondria of thosecells, thereby sparing healthy cells and tissue from the effects of theanalog or derivative. Hence, the agent of the present invention isparticularly suited for treatment for diseases characterized by cellularhyperproliferation. The skilled artisan can readily identify diseasespresenting such activity or alternatively can readily screen the diseaseof interest for sensitivity to such analogs or derivatives.

The alkyl fatty acid analogs or derivatives of the present invention,and pharmaceutical formulations thereof, are expected to be useful insuch general cancer types as carcinoma, sarcoma, lymphoma and leukemia,germ cell tumor, and blastoma. More specifically, the pharmaceuticalcomposition of the present invention is expected to be useful in primaryor metastatic melanoma, lung cancer, liver cancer, Hodgkin's andnon-Hodgkin's lymphoma, uterine cancer, cervical cancer, bladder cancer,kidney cancer, colon cancer, and adenocarcinomas such as breast cancer,prostate cancer, ovarian cancer, and pancreatic cancer, withoutlimitation. Non-limiting examples of other diseases characterized bycellular hyperproliferation amenable to the agent of the presentinvention include age-related macular degeneration; Crohn's disease;cirrhosis; chronic inflammatory-related disorders; diabetic retinopathyor neuropathy; granulomatosis; immune hyperproliferation associated withorgan or tissue transplantation; an immunoproliferative disease ordisorder (e.g., inflammatory bowel disease, psoriasis, rheumatoidarthritis, or systemic lupus erythematosus); vascular hyperproliferationsecondary to retinal hypoxia; or vasculitis.

By adapting the methods described herein, the alkyl fatty acid analogsor derivatives of the present invention, and pharmaceutical formulationsthereof, may also be used in the treatment, prevention, imaging, ordiagnosis of diseases other than those characterized by cellularhyperproliferation. For example, eukaryotic pathogens of humans andother animals are generally much more difficult to treat than bacterialpathogens because eukaryotic cells are so much more similar to animalcells than are bacterial cells. Such eukaryotic pathogens includeprotozoans such as those causing malaria as well as fungal and algalpathogens. Because of the remarkable lack of toxicity of the alkyl fattyacid analogs or derivatives of the present invention to non-transformedhuman and animal cells, and because many eukaryotic pathogens are likelyto pass through life cycle stages in which their PDH, α-KDH, ADH, and/orBCKADH complexes become sensitive to such analogs or derivatives, thealkyl fatty acid analogs or derivatives of the present invention, andpharmaceutical formulations thereof, can be used as bacteriocidalagents.

Specific embodiments of the invention will now be demonstrated byreference to the following examples. It should be understood that theseexamples are disclosed solely by way of illustrating the invention andshould not be taken in any way to limit the scope of the presentinvention.

Example 1 Screening of Analogs for Cell Kill Activity in Cancer CellsObjective

The objective of this investigation was to assess the in vitro cellkilling activities of analogs of lipoic acid in BXPC3 human pancreatic,H460 non small lung carcinoma, and SF539 human gliosarcoma cancer cells.

Materials and Methods Materials

All materials were obtained through normal distribution channels fromthe manufacturer stated.

Costar opaque-walled plate, Corning Costar Corporation, Cambridge,Mass., cat. no. 3917, Fisher Scientific cat no. 07-200-628

FLUOstar OPTIMA, BMG LABTECH, Offenburg, Germany

CellTiter Glo® (CTG) Luminescent Cell Viability Assay, Promega, FisherScientific cat no. PR-G7573

RPMI 1640 Tissue culture medium, Mediatech, Fisher Scientific cat. no.MT-10040-CV

Fetal Bovine Serum (FBS), Fisher Scientific cat. no. MTT35011CV

Penicillin and Steptomycin, Fisher Scientific cat. no. MT 30-009-CI

Tumor Cell Lines

Three human tumor cell types, BXPC3 human pancreatic cancer, H460 nonsmall lung carcinoma, and SF539 human gliosarcoma, were used in thisinvestigation. The BXPC3 and H460 cells were originally obtained fromAmerican Type Cell Culture (ATCC). The SF539 cells were originallyobtained from the NCI AIDS and Cancer Specimen Bank (ACSB). All tumorcells were maintained at 37° C. in a humidified 5% CO₂ atmosphere in T75tissue culture flasks containing 20 mL of Roswell Park MemorialInstitute (RPMI) 1640 containing 2 mM L-glutamine, 10% FBS and 1%penicillin and streptomycin (100 IU/mL penicillin and 100 μg/mLstreptomycin). The tumor cells were split at a ratio of 1:5 every 4-5days by trypsinization and resuspended in fresh medium in a new flask.Cells were harvested for experiments at 70-90% confluency.

Test Articles

Stock solutions of each analog were prepared at a concentration of 200and 100 mM in DMSO. Five μL of this solution was diluted in 10.0 mL of0.5% serum containing RPMI media to give the desired 100 μM and 50 μMsolutions in 0.05% DMSO.

Study Procedures Study Design

The cancer cells were seeded at 4000 cells/well for H460 cells and 6000cells/well for BXPC3 and SF 539 cells and incubated 24 hours. Thekilling activity of analogs was assayed at 50 μM and 100 μMconcentrations. The tumor cells were treated for 24 hours with the testarticle, and after 24 hours of treatment the number of viable tumorcells was determined using the CTG assay.

Cell Seeding for Experiments

Cells were grown to 70-90% confluency, medium was removed, and the cellmonolayers were washed briefly by adding 5 mL of phosphate buffer saline(PBS) followed by aspiration. Trypsin-ethylenediaminetetraacetic acid(EDTA) (4 mL) was added to each flask, and the flask was placed in thetissue culture incubator for 5 minutes. Serum-containing medium (10 mL)was added to halt the enzymatic reactions, and cells were disaggregatedby repeated resuspension with serological pipette. The cell-containingmedium (20 μL) was added to 20 μL of 0.4% Trypan Blue solution, mixed,and 10 μL of this cell-containing mixture was placed in a chamber of thehemocytometer. The number of viable cells was determined by counting thenumber of viable cells (cells that excluded Trypan Blue) in the fourcorner squares of the hemocytometer chamber at 100× magnification, toget the average number of cells present. The volume of cells needed wasdetermined by the following formula:

${{Volume}\mspace{14mu} {of}\mspace{14mu} {cells}\mspace{14mu} {needed}\mspace{14mu} \ldots} = \frac{\# \mspace{14mu} {of}\mspace{14mu} {cells}\mspace{14mu} {need}\mspace{14mu} {for}\mspace{14mu} {the}\mspace{14mu} {assay}\mspace{14mu} ({mL})}{\# \mspace{14mu} {of}\mspace{14mu} {cells}\mspace{14mu} {counted}\mspace{14mu} ({mL})}$

where # of cells counted (mL)=average # of cells on hemocytometer×2(dilution factor)×10⁴.

The number of cells targeted for the study is 4×10³ per well for H460cells and 6×10³ per well for BXPC3 and SF539 cells in 100 μL of medium.The actual number of cells were counted and seeded in the wells of a 96well-plate. The cells were incubated for approximately 24 hours beforeaddition of test article.

Treatment with Test Article

The media in the plate was removed by aspiration, and 100 μL of the testarticle at a final concentration of 50 μM or 100 μM was added to thecells. After exposure to the test articles for 24 hours, the number ofviable cells in each well was determined and the percent of viable cellsrelative to control (in the absence of test article) were calculated.Additionally, a set of wells was treated with cell culture medium in theabsence of cells to obtain a value for background luminescence. Aseparate set of cells was seeded at the same time in a clear 96-wellplate and observed under the microscope at 24 hours, following additionof the test article to estimate the amount of cells present aftertreatment.

Determination of the Number of Viable Cells by the CTG Assay

The number of viable cells was determined by using the CTG assay.Specifically, reagents were mixed and allowed to come to roomtemperature according to instructions from Promega, Inc. (Madison,Wis.). Cell plates were removed from the cell culture incubator and lefton the bench for thirty minutes until they reached room temperature.1004, per well of CTG reagent was added with the 12-channel Eppendorfpipettor. The cells were lysed by shaking the plate for two minutes in ashaker. The cells were kept in room temperature for ten minutes tostabilize the luminescent signal. The luminescence was measured usingthe FLUOstar OPTIMA plate reader (BMG Labtech, Inc., Durham, N.C.).

Calculation of Cell Killing Activity

Data from luminescence readings was copied onto EXCEL spreadsheets, andcell growth relative to untreated cells was calculated, using thefollowing equation:

${\% \mspace{14mu} {growth}\mspace{14mu} {related}\mspace{14mu} {to}\mspace{14mu} {NT}} = {\frac{{mean}\mspace{14mu} {luminescence}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {test}\mspace{14mu} {article}}{{mean}\mspace{14mu} {luminescence}\mspace{14mu} {untreated}} \times 100\%}$

RESULTS AND CONCLUSION

The results of the experiment are summarized in Table 1.

TABLE 1 Comparison of in vitro cancer cell killing activity of analogsof the present invention % Viable Cells Remaining (0.5% serum and 0.05%DMSO) BXPC3 H460 SF539 % avg % avg % avg live % avg live live % avg livelive % avg live cells @ cells @ cells @ cells @ cells @ cells @ Article50 μM 100 μM 50 μM 100 μM 50 μM 100 μM A 84.0 75.0 89.0 70.0 100.0 98.0B 84.0 91.0 97.0 67.0 99.0 99.0 C 70.9 36.1 69.8 27.1 74.9 23.2 D 0.33.9 4.1 2.7 2.1 0.1 E 0.0 0.0 0.0 0.3 0.0 0.0 F 2.0 3.9 2.4 4.0 0.0 0.0G 99.7 41.7 101.7 37.6 91.6 30.3

As is evident from Table 1, each of the analogs of the present inventiondemonstrated in vitro cell killing activity against at least one of thecancer cell lines tested at either the 50 μM concentration, the 100 μMconcentration, or both.

The foregoing discussion discloses and describes merely exemplaryembodiments of the present invention. One skilled in the art willreadily recognize from such discussion, and from the accompanyingclaims, that various changes, modifications and variations can be madetherein without departing from the spirit and scope of the invention asdefined in the following claims. Furthermore, while exemplaryembodiments have been expressed herein, others practiced in the art maybe aware of other designs or uses of the present invention. Thus, whilethe present invention has been described in connection with exemplaryembodiments thereof, it will be understood that many modifications inboth design and use will be apparent to those of ordinary skill in theart, and this application is intended to cover any adaptations orvariations thereof. It is therefore manifestly intended that thisinvention be limited only by the claims and the equivalents thereof.Additionally, all patent applications, patents, and other publicationscited herein are incorporated by reference in their entirety.

The invention to be claimed is:
 1. An analog or derivative of an alkylfatty acid having the general formula:

wherein n is 1-2 and x is 1-16, with the resulting hydrocarbon chainpotentially being mixed saturated or unsaturated; wherein R₁ and R₂ areindependently hydrogen, alkyl, alkenyl, alkynyl, alkylaryl, heteroaryl,or alkylheteroaryl; wherein R₃ and R₄ are independently a thioether, athioester, an ether, an ester, an amine, an amide, a rare earth metalsuch as but not limited to gadolinium, a transition metal such as butnot limited to platinum or indium, or a nonmetal such as but not limitedto selenium; wherein R₅ is hydrogen, nitrogen, alkyl, alkenyl, alkynyl,alkylaryl, heteroaryl, alkylheteroaryl, an ether, an amine, aphosphonium, a phosphonate, or a sulfonic acid; and salts, prodrugs, orsolvates thereof.
 2. The analog or derivative of claim 3, wherein theanalog or derivative is selected from the group consisting of:


3. A pharmaceutical formulation comprising a therapeutically-effectiveamount of at least one alkyl fatty acid analog or derivative of claim 1and at least one pharmaceutically-acceptable carrier or excipienttherefor.
 4. The pharmaceutical formulation of claim 3, wherein the atleast one alkyl fatty acid analog or derivative is present in an amountto provide from about 0.001 mg/m² to about 10 g/m².
 5. A method oftreating, preventing, imaging, or diagnosing a disease characterized bydiseased cells or tissues that are sensitive to alkyl fatty acid analogsor derivatives comprising administering to a patient in need thereof atherapeutically-effective amount of at least one alkyl fatty acid analogor derivative according to claim
 1. 6. The method of claim 5, whereinthe at least one alkyl fatty acid analog or derivative is in apharmaceutical formulation further comprising at least onepharmaceutically-acceptable additive.